Clostridium is a genus of anaerobic Gram-positive spore-forming bacterium that is implicated in the decomposition and degradation of organic matters in soil and the anaerobic compartments of mammalian gastrointestinal tracts. To affect their function, many Clostridium spp. encode and express degradative enzymes targeting various components of living matter. Some of these enzymes have evolved into highly potent toxins and cytotoxins. The expression of these toxins can turn some Clostridium species into opportunistic pathogens, including Clostridium tetani, Clostridium botulinum, Clostridium perfringens and Clostridium difficile, which cause tetanus, botulism, gas gangrene, and antibiotic-associated colitis, respectively.
C. difficile infection (CDI) is one of the most common nosocomial infections in industrialized nations, often prevalent in hospital patients undergoing antibiotic therapy due to the disruption of normal colonic flora caused by the antibacterial agent(s). There are an estimated 500,000 CDI cases in the United States each year (Campbell et al., Infect Control Hosp Epidemiol, 30(6): 526-33 (2009); Rupnik et al., Nature Reviews, 7: 527-36 (2009)). The number of C. difficile cases reported in the United States has risen, as noted by an increase of 92% from 2001 to 2005 in the rate of hospitalizations with CDI discharge diagnoses. Almost all antibiotics, including clindamycin, cephalosporins, penicillins, and fluoroquinolones, have been associated with symptomatic disease caused by toxigenic C. difficile (Bartlett, J. G., Clin Infect Dis. 46(10): 1489-92 (2008); Gorbach, S. L., N Engl J Med, 341(22): 1690-1 (1999); Kelly et al., N Engl J Med 330(4): 257-62 (1994)). C. difficile-associated disease (CDAD) includes antibiotic-associated diarrhea, colitis and pseudomembranous colitis and in some cases, may progress to toxic megacolon, sepsis and death. For this reason, CDI is a major healthcare concern. Although some patients suffering from CDAD respond to cessation of antibiotic therapy, most require treatment with further agents, such as metronidazole or vancomycin (reviewed in DuPont et al., Current Opinion in Infectious Diseases 21:500-507 (2008)). Existing therapies often fail to eliminate CDAD or lead to recurrent illness; thus, new methods of prophylactic or therapeutic treatment are needed.
Over the last decade, increases in severe or fatal infections, standard of care therapy failures, emergence of a more virulent, epidemic strain (NAP1/027/BI), increasing community acquired infection, and the incidence of recurrent infection (estimated to be ˜15-25%) have contributed to an increased necessity for therapeutic and/or prophylactic treatments for CDI (Aslam et al., Lancet, 365(9469): 1464 (2005); Loo, et al., N Engl J Med, 365(18): 1693-703 (2006); McDonald et al., N Engl J Med, 353(23): 2433-41 (2005); Muto, C. A., Infect Control Hosp Epidemiol, 26(1): 10-2 (2005); Pépin et al., Cmaj., 171(5): 466-72 (2004); Zar, et al., Clin Infect Dis, 45(3): 302-7. (2007)). Several studies have recently suggested that the incidence of community acquired CDI has increased (MMWR Morb Mortal Wkly Rep, 57(13): 340-3 (2008); Hirschhorn et al., J Infect Dis., 169(1): 127-33 (1994); Karlstrom et al., Clin Infect Dis, 26(1): 141-5 (1998); Kyne et al., J Infect, 36(3): 287-8 (1998); Lambert et al., Infect Control Hosp Epidemiol, 30(10): 945-51 (2009); Noren et al., J Clin Microbiol, 42(8): p. 3635-43 (2004); Paltansing et al., Clin Microbiol Infect, 13(11): 1058-64 (2007).
Toxigenic isolates of Clostridium difficile usually encode two large clostridial toxins, referred to as toxin A and toxin B (“TcdA” and “TcdB”), which are part of a larger family of toxins called large Clostridial toxins (LCT), due to their unusual large molecular weight (>250 kDa) and mechanism of action (Schirmer and Aktories, Biochimica et Biophysica Acta 1673: 66-74 (2004)). Additional toxigenic strains express only toxin B and not toxin A. TcdA and TcdB have been demonstrated to be the sole causative agents of CDI. A subset of C. difficile strains also produce an actin-ADP-ribosylating toxin, referred to as C. difficile transferase, or CDT. This toxin, which is commonly referred to as binary toxin, is particularly associated with highly virulent, epidemic strains such as NAP1/027/BI and consists of the two proteins CDTa and CDTb.
The CDTb subunit forms a heptameric ring-like structure which permits entry into the cytoplasm of the enzymatically active, CDTa protein. The CDTa protein catalyzes the ADP-ribosylation of G-actin at Arginine 177 and prevents actin polymerization (Barth, H., et al., Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins. Microbiol Mol Biol Rev, 68(3): 373-402 (2004)). The precise role of binary toxin in the pathogenesis of C. difficile disease is poorly defined; however, these toxins have been shown to function as enterotoxins (Geric, B., et al., Journal of Infectious Diseases 193(8): 1143-1150 (2006)) to induce the formation of microtubules and, thereby increase C. difficile colonization (Schwan, C., et al., PLoS Pathog, 5(10): e1000626 (2009)), and have been more recently associated with increased mortality compared to non-binary toxin containing strains (Bacci et al., Emerg. Infect. Dis., 17(6): 976-82 (2011); Goldenberg S D, Journal of Infection, 62: 355-62 (2011)).
Several approaches to C. difficile vaccine development are described in the literature, including passive immunization with C. difficile toxin-neutralizing polyclonal serum (see Thomas et al., WO 99/20304 and WO 2005/058353), immunization with toxin fragments (Ward et al., WO 98/59053; Lyerly et al., Current Microbiology 21: 29-32 (1990); WO 00/061761) and immunization with formalin-inactivated fully native toxin (Torres et al., Infection and Immunity 63(12): 4619-4627 (1995); Kim et al., Infection and Immunity 55(12): 2984-2992(1987)); Fernie et al. Devel. Biol. Standard 53: 325-32 (1983); Libby et al., Infect Immun 36: 822-9 (1982); Ghose et al., Infection and Immunity 75(6): 2826-2832). Also described are DNA-based vaccines encoding toxin fragments (Gardiner et al., Vaccine 27:3598-3604 (2009)), including binary toxin (J. E. Galen, WO 2011/060431). Ellingsworth et al. (WO 2012/028741) disclose a recombinant fusion protein comprising the receptor binding domains of TcdA and TcdB.
Also described is the use of recombinant toxin-based vaccines in which specific sites that contribute to toxicity are mutated. Ballard and Spyres describe a recombinant TcdB vaccine in which the cysteine at position 395 is replaced with another amino acid (U.S. Pat. No. 7,226,597). Other sites described as important to enzyme activity are the tryptophan residue at position 102 (W102) of TcdB and the catalytic triad motif DXD which is a signature motif of the glycosyltransferase A family, of which LCTs are part (Busch et al., J Biol. Chem. 275(18): 13228-234 (2000)). Feng et al. (WO 2011/068953) describe TcdA toxins with single, double, or triple mutations and a TcdB toxin with double mutations to eliminate toxicity.